The eutrophication of freshwater environments caused by an excess inflow of phosphorus has become a serious environmental issue because it is a crucial factor for the occurrence of harmful algal blooms (HABs) in essential water resources. The adsorptive removal of phosphorus from discharged phosphorus containing effluents has been recognized as one of the most promising solutions in the prevention of eutrophication. In the present study, a polyvinyl chloride (PVC)-polyethyleneimine (PEI) composite fiber (PEI-PVC) was suggested as a stable and recoverable adsorbent for the removal of phosphorus from aqueous phases. The newly introduced amine groups of the PEI-PVC were confirmed by a comparison between the FT-IR and XPS results of the PVC and PEI-PVC. The phosphorus sorption on the PEI-PVC was pH dependent. At the optimum pH for phosphorus adsorption (pH 5), the maximum adsorption capacity of the PEI-PVC fiber was estimated to be 11.2 times higher (19.66 ± 0.82 mg/g) than that of conventional activated carbon (1.75 ± 0.4 mg/g) using the Langmuir isotherm model. The phosphorus adsorption equilibrium of the PEI-PVC was reached within 30 min at pH 5. From the phosphorus-loaded PEI-PVC, 97.4% of the adsorbed amount of phosphorus on the PEI-PVC could be recovered by employing a desorption process using 1M HCl solution without sorbent destruction. The regenerated PEI-PVC through the desorption process maintained a phosphorus sorption capacity almost equal to that of the first use. In addition, consistently with the PVC fiber, the PEI-PVC fiber did not elute any toxic chlorines into the solution during light irradiation. Based on these results, the PEI-PVC fiber can be suggested as a feasible and stable adsorbent for phosphorus removal. Phosphorus (P) is widely applied as an important element in various areas of human activity  . AccordiVarious water treatments based on physiochemical and biological technologies, such as electrodialysis, struvite precipitation, membrane filtration, and biological digestion, are being developed and applied to remove phosphorus from aqueous phases ,7,8,9,102 and ZrO2, have been reported to have considerable phosphate adsorption capacities at 16.3 and 47.4 mg/g, respectively [Adsorption technology is considered as a reliable and economical application method for the removal of phosphorus from aqueous solutions, because it does not require any additional separate operation processes, additives, or power sources . Therefoectively . Clay miectively  and clinectively , have beectively . Howeverectively . Therefo2PO4−, HPO42−, and PO43−) in aqueous phases depending on its pKa property, it can be electrostatically adsorbed to cationic functional groups such as amine groups. However, the chemical structure of PVC does not possess any functional groups as adsorption sites for anionic phosphates. To aminate the PVC matrix, amine-rich ionic polymer polyethyleneimine (PEI) was applied because PEI modification is a well-known method that can efficiently enhance the adsorption performance of adsorbents for anionic pollutants, including phosphates [In the present study, to fabricate an easily recoverable and highly stable adsorbent for the adsorptive removal of phosphorus, the synthetic polymer polyvinyl chloride (PVC), which is a chemically/physically stable, inexpensive, and light-weight commercial thermoplastic , was apposphates ,20. Geneosphates . Consequ2HPO4, >98%) were purchased from Sigma-Aldrich Korea Ltd. . N,N-dimethylformamide  was supplied by Duksan Science . HCl (36%) and NaOH (>97%) were purchased from Samchun Chemicals Co., Ltd.  and Daejung Chemicals & Metals Co., Ltd. , respectively.Polyvinyl chloride , branched polyethyleneimine (PEI) solution , and potassium phosphate dibasic (Kv/v in D.W.) to form a fiber sorbent using a plastic hub needle with air compression. The fabricated PEI-PVC fibers were washed with distilled water several times for the removal of residual DMF and other impurities. The prepared PEI-PVC fiber was then freeze-dried for 24 h. To minimize the effect of humidity and CO2 on the sorbent, it was stored in a desiccator during period adsorption tests.To prepare the PEI composite PVC fiber sorbent (PEI-PVC), two grams of PEI solution (50% in solution) was mixed with 30 mL of PVC solution (2 g PVC/30 mL DMF). Next, the mixture was allowed to react at 80 °C for 4 h in a water bath. The reacted mixture was continuously injected into a methanol solution  mode with scanning number of 100 and 0.9 cm−1 scanning resolution. Furthermore, to analyze minute changes in the functional groups of the sorbents, the XPS signals for N1s and Cl2p of the PVC and PEI-PVC were analyzed via XPS .The measurement of functional group characteristics on the sorbents was carried out using Fourier transform infrared spectroscopy . FT-IR analyses of adsorbents were conducted within the wavenumber range of 650–4000 cmTo determine and compare the phosphorus adsorption performances of the PEI-PVC and AC, pH effect (pH edge), kinetics, and isotherm tests were performed in a batch system. In the adsorption experiments, the phosphorus stock solution  was diluted with distilled water to prepare test solutions containing desired initial concentration of phosphorus. For the adsorption test, the adsorbents (adsorbent dosage: 1 g/L) were agitated with the prepared phosphorus solutions in a shaking incubator until reaching the adsorption equilibrium state at 170 rpm and 25 °C. In addition, during the adsorption process, the solution pH was continuously monitored and adjusted to the desired pH range using HCl (0.1 M and 1 M) and NaOH (0.1 M and 1 M) solutions. The detailed conditions of each experiment are summarized in q (mg/g) is the phosphorus adsorption capacity of the adsorbent. M (g) is the applied adsorbent weight to the adsorption experiment. iC and fC are the phosphorus concentrations (mg/L) at the initial and final adsorption states, respectively. iV (L) is the initial volume of the sample, and fV (L) is the final volume considering the amount of added acid/alkaline solution for pH adjustment.After adsorption processes, to measure residual phosphorus concentration in the experimental samples, liquid–solid separation was conducted using high-speed centrifugation at 9000 rpm. The residual phosphorus concentration in the supernatants was detected using inductively coupled plasma-optical emission spectrometry . The adsorbed amount of phosphorus by the adsorbents could be calculated using Equation (1) as presented below:To determine adsorbent reusability of the used PEI-PVC fiber in phosphorus adsorption, phosphorus-loaded PEI-PVC fiber was prepared through adsorption process (agitating 0.03 g of PEI-PVC fiber with 30 mL of phosphorus solution for 4 h at pH 5 and 25 °C). The initial phosphorus concentration was 98.25 ± 0.30 mg/L. After the sorption process, phosphorus-loaded PEI-PVC was separated from the mixture and briefly rinsed using 30 mL of distilled water one time to remove residual phosphorus solution. Then, the rinsed phosphorus-loaded PEI-PVC was treated in 30 mL of 1M HCl solution for 4 h to dissociate the adsorbed phosphorus from used PEI-PVC. After that, the regenerated PEI-PVC was washed several times using distilled water to remove the remaining HCl solution. The adsorption process was performed again using completely regenerated PEI-PVC under the same conditions as the first adsorption trial. The adsorption and desorption processes using PEI-PVC were repeated in three cycles.2) at 25 °C. The eluted chlorine concentration in the gathered samples was measured using a residual chlorine meter  following the manufacturer’s protocol. All experiments were conducted thrice.To measure the eluted chlorine levels that were generated from the PVC backbone of the sorbents, PVC and PEI-PVC fibers were agitated in 150 mL of autoclaved distilled water for 24 h under white fluorescent light , 1324 (C–H deforming in plane), 1093 (C–C stretching), and 970 cm−1 (CH2 rocking) indicate the chemical bond properties of the PVC matrix [−1 and the strong peak at 1654 cm−1 are attributed to the asymmetric N–H stretching of amines and the bending of secondary amines, respectively [−1 can be attributed to –NH bending or C–N stretching [−1 assigned to the bending bond of C–H near Cl in the PVC backbone decreased and shifted to 1235 cm−1 in the FT-IR spectra of the PEI-PVC. These observed peak changes in the FT-IR result of the PEI-PVC fiber might indicate increased amine groups and decreased Cl amounts in the PEI-PVC fiber due to a nucleophilic substitute reaction between the chloro-groups of the PVC backbone and the amine groups of the PEI molecules [1s) and chloride (XPS Cl2p) atoms of the PVC-based fibers . However, in the case of the PEI-PVC, the strong N1s signal was newly observed in the binding energy range of 392–404 eV. This signal might be attributed to the amine groups of the coupled PEI molecules with the PVC backbone since N1s peaks of –NH2 and C–N = C bonds in the PEI molecules could be observed at 398.8 and 399.5 eV [2p signals, a higher XPS signal intensity was observed in the XPS result of the PVC than in that of the PEI-PVC. This evidently indicates that the amount of chloride was decreased in the PEI-PVC fiber compared to that of the PVC fiber owing to the substitution reaction between the PEI molecules and the chloride in the PVC.Since the adsorption target phosphorus usually exhibits anionic phosphate forms in an aqueous solution depending on its pKolecules ,23. It hmolecule . In addiC matrix . In the ectively . In addiretching ,27. Furtolecules . Additiod fibers . When co399.5 eV . This re2p results, since the amount of chloride in the PEI-PVC was reduced, it was expected that the elution of chlorines from the PEI-PVC fiber might be reduced compared to that of the PVC. Therefore, the generated chlorines from the PVC and PEI-PVC fibers were compared  in PEI molecules can be observed at approximately pH 4.5, 6.7, and 11.6, respectively [3PO4) and electrostatically adsorbable phosphate anions  depending on the solution pH condition in the aqueous solution [2PO4− at pH 2. However, the almost amount of phosphorus can be changed to the phosphate anionic form at pH 3.5. The increasing fraction of anionic phosphate might contribute to the increasing phosphorus uptake of the PEI-PVC in the range of pH 2–pH 3.5 by the enhancement of the electrostatic attraction between the PEI-PVC and phosphate. Above pH 5, although the almost amount of phosphorus can be formed as the adsorbable phosphate anions, since the positivity of the PEI-PVC could be lost by the deprotonation of amine groups depending on their pKa properties, the phosphorus sorption capacity of the PEI-PVC might be reduced. The conventional adsorbent, AC, did not show pH dependence on phosphorus adsorption capacity. However, the phosphorus sorption capacities of the AC were significantly lower than those of the PEI-PVC fiber under all of the pH conditions.ectively . Accordisolution . Accordi3PO4), adsorbed phosphorus might be recovered from phosphate-loaded PEI-PVC. To estimate the desorbed amount of phosphorus, the phosphate-adsorbed PEI-PVC at pH 5 was treated using 1M HCl solution. The pH-dependence property of the PEI-PVC fiber on phosphorus adsorption could be applied to the phosphorus desorption process for the reuse of the PEI-PVC. As discussed above, under highly acidic conditions, since phosphate anions can be changed to uncharged phosphoric acid (Heq and tq are the phosphorus sorption capacity of the sorbents (mg/g) at the sorption equilibrium state and specific sorption reaction time (t), respectively. The parameter k2 (g/mg min) indicates the estimated adsorption rate constants from the pseudo-second-order model, respectively.Adsorption equilibrium time is an important factor to determine the efficiency of the developed sorbent for the target materials. Therefore, to determine the sorption equilibrium time of the PEI-PVC for phosphorus, a kinetic experiment using the PEI-PVC was performed in a batch system at pH 5. From the kinetic result , the PEI2) of the pseudo-second-order model for the phosphorus sorption kinetic data of the PEI-PVC and AC were 0.9585 and 0.9211, respectively. These high R2 values indicate that the phosphorus sorption kinetic experimental results of the PEI-PVC and AC were predicted well by the pseudo-second-order kinetic model. The sorption reaction rate (k2) of the AC was estimated as 0.037 ± 0.013 g/mg·min using the kinetic model. This was a value approximately 6.5 times higher than that of the PEI-PVC (0.0057 ± 0.0007 g/mg·min), and it was better connected to the faster sorption equilibrium state of the AC than the PEI-PVC fiber. However, as shown in the experimental kinetic results, the PEI-PVC showed a comparable sorption equilibrium time to that of the AC. Furthermore, the phosphorus sorption capacity (qe) calculated from the sorption kinetic data of the PEI-PVC was 5.9 times higher (23.39 ± 0.47 mg/g) than that of the AC (3.94 ± 0.21 mg/g) using the pseudo-second-order model. Based on the kinetics result, the PEI-PVC fiber can be suggested as an efficient substitute for conventional adsorbents  to rapidly counteract the inflow of phosphorus from the effluents of point or non-point pollutant sources.The estimated kinetic parameters for the PEI-PVC and AC from the pseudo-second-order kinetic model are summarized in max) of the sorbent toward the target pollutant is an essential factor for the evaluation of the adsorption performance of the developed adsorbent. Therefore, to determine the qmax of the developed sorbent PEI-PVC fiber, isotherm experiments were performed under various pH conditions (pH 5–7). In addition, the maximum phosphorus  sorption capacities of the PEI-PVC fiber were compared with those of the conventional adsorbent (AC). The obtained isotherm results using the PEI-PVC and AC at different pH values are presented in eq is the experimentally calculated phosphorus sorption capacity (mg/g). fC (mg/L) is the final phosphorus concentration in the solution after the adsorption process. The parameters fK (L/g) and n are the Freundlich constants, which can be indicators of sorption capacity and sorption intensity, respectively. The parameters in the Langmuir model equation max,Lq and b indicate the maximum capacity (mg/g) and sorption affinity (L/mg) of the adsorbent for phosphorus, respectively.Together with the sorption kinetic property, the maximum sorption capacity (qfK) associated with the phosphorus adsorption capacity of the PEI-PVC fiber increased with the decrease in the solution pH from pH 7 (5.12 ± 0.97 L/g) to pH 5 (9.83 ± 1.34 L/g). The dimensionless n value of the PEI-PVC in phosphorus adsorption showed a similar tendency to that of the fK values. The recorded n values from the isotherm data of the PEI-PVC fiber for phosphorus at pH 5, pH 6, and pH 7 were 5.21 ± 1.05, 5.29 ± 1.10, and 4.14 ± 0.91, respectively. According to Toor and Jin [2 values: 0.9755, 0.9843, and 0.9844 at pH 5, 6, and 7, respectively), than those of the Freundlich model  at all solution pH values. The maximum phosphorus sorption capacities  of the PEI-PVC fiber were estimated from the Langmuir model as 19.66 ± 0.82, 16.54 ± 0.54, and 13.66 ± 0.51 mg/g at pH 5, 6, and 7, respectively. In addition, the isotherm result of the PEI-PVC fiber showed a higher b value at pH 5 (2.08 ± 0.59 L/mg) than at pH 6 (1.65 ± 0.35 L/mg) and pH 7 (0.42 ± 0.08 L/mg) using the Langmuir model. As discussed above section, since the higher amount of amine groups in the PEI-PVC fiber could be positively activated at pH 5 compared to those at pH 6 and pH 7 depending on the pKa properties of the amine groups, the higher values of the Freundlich and Langmuir isotherm parameters corresponding to the sorption capacity and affinity for phosphorus adsorption could be calculated from the isotherm data.As summarized in  and Jin , if the maxq values than those of the PEI-PVC fiber at natural pH. For instance, the qmax values for the phosphorus of Zr-loaded orange waste gel, Zr-loaded MUROMAC, and Zr-loaded okara were calculated as 57, 43, and 14.39 mg/g, respectively. Under the condition of pH 3, the covalently cross-linked chitosan by epichlorohydrin showed higher phosphorus adsorption performance (38.22 mg/g) compared to the PEI-PVC fiber (approximately 27 mg/g), whereas the maximum phosphorus uptake of electrostatically cross-linked chitosan by sodium citrate (13.3 mg/g) was smaller than that of the PEI-PVC. Although we could find several adsorbents showing higher qmax values for phosphorus than the PEI-PVC fiber, there still remains an opportunity to enhance the phosphorus adsorption performance of the PEI-PVC fiber through the optimization of adsorbent processes and the additional application of adsorption-supporting materials. In addition, as shown in Furthermore, a comparison of the maximum phosphorus adsorption capacity of the PEI-PVC fiber with that of previously reported adsorbents was performed . As show1s results of the prepared PEI-PVC, the aminated characteristics of the PEI-PVC fiber were well determined. The adsorption of phosphorus on the PEI-PVC was affected by the solution pH. The maximum phosphorus sorption capacity (qmax) of the PEI-PVC fiber was estimated as 19.66 ± 0.82 mg/g at pH 5 using the Langmuir equation. At pH 6 and pH 7, the qmax values of the PEI-PVC fiber were calculated as 16.54 ± 0.54 and 13.66 ± 0.51, respectively. In the kinetic analysis, the PEI-PVC showed a rapid phosphorus adsorption equilibrium time within 30 min at pH 5. The used PEI-PVC could be regenerated without destructing the adsorbent matrix using the 1M HCl solution by the desorption of the adsorbed phosphorus in the PEI-PVC fiber (desorption rate: >98%). In addition, the regenerated PEI-PVC was reusable for phosphorus adsorption without the loss of sorption capacity. Furthermore, it was found that the PEI-PVC fiber did not discharge toxic chlorines during light irradiation. Based on the results, we can suggest that the stable and reusable PEI-PVC fiber can be a feasible adsorbent for phosphorus removal from aqueous phases. However, to further enhance its phosphorus adsorption performance, additional research on the optimization of adsorbent processes and the additional application of adsorption-supporting materials might be needed.In the present study, the amination of a non-functional PVC matrix was performed using a simple chemical reaction process between PEI and PVC to develop an adsorbent for phosphorus removal from aqueous phases. From the FT-IR and XPS-N


Currently, more than 30,200,000 COVID-19 cases have been diagnosed in Germany alone. However, data regarding prevalence of COVID-19 in children, both in Germany and internationally, are sparse. We sought to evaluate the number of infected children by measuring IgG antibodies.recomLine Blot) in 365 German children aged 3–16 years from 30 schools and preschools. We used multiple serological tests because the filter paper test Euroimmun® ELISA performs better in terms of sensitivity and specificity than the rapid test NADAL®. The Roche® ECLIA test is used to detect SARS-CoV-2 spike protein, and the recomLine Blot test is used to rule out the possibility of infection by seasonal SARS-viruses and to test for specific SARS-CoV-2 proteins . In addition, one parent each (n = 336), and 4–5 teachers/caregivers (n = 90) per institution were tested for IgG antibodies from capillary blood samples. The total study duration was 4 months per child, including the first follow-up after 2 months and the second after 4 months.Oropharyngeal swabs were collected between December 2020 and August 2021 to measure SARS-CoV-2, and capillary blood for the detection of SARS-CoV-2 antibodies (by rapid test NADAL® and filter paper test Euroimmun® ELISA); venous blood was taken for validation  were positive for SARS-CoV-2 IgG antibodies using Euroimmun® ELISA. Seven children reported previously testing positive for SARS-CoV-2; each of these was confirmed by the Roche® Anti-SARS-CoV-2-ECLIA (antibody to spike protein 1) test. SARS-CoV-2 IgG antibodies persisted over a 4-month period, but levels decreased significantly (p = 0.004) within this timeframe. The median IgG values were 192.0 BAU/ml , 123.6 BAU/ml  and 89.9 BAU/ml  at baseline, 2 months and 4 months after baseline, respectively. During the study period, no child tested positive for SARS-CoV-2 by oropharyngeal swab. A total of 4.3% of all parents and 3.7% of teachers/caregivers tested positive for IgG antibodies by Euroimmun® ELISA at baseline.We noted a rather low seroprevalence in children despite an under-reporting of SARS-CoV-2 infections. Measurement of IgG antibodies derived from capillary blood appears to be a valid tool to detect asymptomatic infections in children. However, no asymptomatic active infection was detected during the study period of 4 months in the whole cohort. Further data on SARS-CoV-2 infections in children are needed, especially in the group of <5-year-olds, as there is currently no licensed vaccine for this age group in Germany. The Robert Koch Institute’s Standing Commission on Vaccination (STIKO) recommended COVID-19 vaccination for 12–17 and 5–11 year olds in August 2021 and May 2022 respectively. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19), which has caused more than 570,000,000 cases of infection as well as more than 6,300,000 deaths worldwide until now (July 2022) . RegardiSero-epidemiological studies have been initiated to collect more detailed data regarding infection events in children. In these studies, SARS-CoV-2-specific IgG antibodies were measured. This is particularly useful because children suffering from COVID-19 are mostly asymptomatic or only mildly infected, drawing little medical attention in comparison to adults –7. As a So far, particularly in children, data is not sufficient to determine how long SARS-CoV-2 antibodies may be detectable in blood. New data from southwest Germany demonstrate that 33.8% of a cohort of SARS-CoV-2 exposed children, aged 6–13 years, were seropositive and that 96.2% of them remained seropositive 11–12 months post infection [Since the current data is inconclusive, more information is required on seroprevalence in children and adolescents considering the COVID-19 pandemic is still ongoing. This may provide a better basis for decision-making on measures for childcare centers and schools to further control the COVID-19 pandemic.To address this issue, we performed the prospective “Augsburg Plus”-Study at the University Hospital Augsburg. The aim of the study was to address the following objectives: 1) To determine the level of antibody immune response to SARS-CoV-2 among preschool- and school-children (age-range 3–16) as well as parents, teachers, and educators 2). To evaluate the infection dynamic over a period of six months.This was a prospective study on the infection dynamics of SARS-CoV-2 infections in children conducted by testing for SARS-CoV-2-transcription-mediated amplification (TMA) and SARS-CoV-2 IgG antibodies in serum over several points in time. The study was conducted at the University Hospital Augsburg in Bavaria (Germany). Altogether 18 childcare facilities, 6 elementary schools, 4 secondary schools and 2 schools with both school types (elementary and secondary school)—a total of 30 facilities within the district and city of Augsburg—cooperated with the Augsburg Plus Study. The institutions were recruited via an initial letter from the Augsburg school authorities for primary and secondary schools, diocese and the district office responsible for preschools, respectively. Since there was initially an insufficient response from the institutions, the sponsors and facility managers were again contacted by phone or e-mail to ask them to collaborate. Children who attended one of the collaborating facilities were enrolled voluntarily and randomly in the study if their parents agreed. The investigations proceeded from December 2020 to August 2021. It was planned to invite each participant to three examinations . In the end, the actual follow-up intervals between appointments were shorter than planned due to an initial low level of enrollments, and were a median length of 2 months each.Children from the collaborating institutions aged 3–16 years and one of their parents were included in the study. Parents had no limitation in age. The aim was to screen 40 children per facility. In addition, about 4 educators or teachers from each of the institutions could register for participation. Each participant had to agree in writing to voluntary participation. An additional consent form was prepared for the children who could already read, in order to inform the children as best as possible. The written informed consent forms for the children were administered by the parents. Each participant was informed that the examinations could only take place if the participants were free from COVID-19 symptoms. If this was the case, the appointed examination date was scheduled. This was applied to each examination appointment.An oropharyngeal swab and a capillary blood sample were taken from every child at each examination time point. The swab was used to determine if the child was acutely infected with SARS-CoV-2. Capillary blood was tested for SARS-CoV-2-IgG antibodies using a rapid test (dichotomous outcome) and a dried blood spot test (quantitative outcome). If either the rapid test or the dried blood spot (Euroimmun® ELISA) test resulted in a positive outcome for IgG antibodies, the child’s venous blood was drawn additionally for a more differential screening (n = 21) . ParentsAt each examination, every participant received a questionnaire. The questionnaire for children and their parents contained questions about COVID-19 symptoms, social contacts, travelling abroad, hygienic behavior, rheumatologic diseases, and medication. In addition, parents were asked about their work situation. Parents were left to decide whether they completed the questionnaire for their child or children, with their child or children, or had the child or children complete it on his/ her or their own. These options were also recorded in each questionnaire. The questionnaires and consent forms were written in the most child-friendly language possible. Teachers and educators received the same questionnaire as parents. Each facility was given a questionnaire to assess what measures they took to reduce the transmission of SARS-CoV-2.The data collection procedures were performed in accordance with the Declaration of Helsinki. Each participant gave written informed consent to participate in the study. The study was approved by the Ethics Committee of the Ludwig-Maximilians-Universität München (Number of the ethics vote: 20–0419).50/ml).Oropharyngeal Swabs (Sigma-Transwab®) were collected and tested on the same day by transcription mediated amplification  showing qualitative lines for IgG and IgM. We focused on the presence or absence of IgG only, ignoring possible IgM-lines. According to the manufacturer, diagnostic sensitivity is 85.2% for IgG 15 to 68 days after infection with SARS-CoV-2, and 60.6% within the time slot of 0 to 14 days. Diagnostic specificity is stated with 99.2% for the combination of IgM and IgG. This test detects antibodies to spike proteins.All children donated a few additional capillary blood drops taken from their finger pads, which were soaked on a filter paper (Euroimmun®). The filter paper was stored in plastic bags for up to 14 days at 2–8°C and then stamped out with a Euroimmun® cutter to achieve exact amounts of dried blood spots. These dried blood spots were diluted and then measured quantitatively with the Euroimmun® Anti-SARS-CoV-2-QuantiVac-ELISA (enzyme-linked immunosorbent assay) (IgG). The ELISA reacts only to antibodies against spike protein and ignores antibodies to the nucleocapsid protein. The accordance of dried blood spots results to venous blood results is specified as 100% by Euroimmun®. Diagnostic sensitivity is determined to be 90.3% at least 10 days after infection and 93.2% for more than 20 days after infection with SARS-CoV-2. Specificity is reported to be 99.8%. Thus, two different antibody-screening tests were our standard for the detection of a past SARS-CoV-2 infection in children.If either the rapid antibody test or the ELISA was positive for SARS-CoV-2 IgG in children, a venous blood sample was taken from the child and tested with an additional CoV-2-antibody-ECLIA (Roche®: Elecsys Anti-SARS-CoV-2 S) the following day as well as a Coronavirus Line Blot (Mikrogen®). The Roche® Anti-SARS-CoV-2-ECLIA detects IgG antibodies to the spike protein. It shows a sensitivity of 98.8% 14 or more days after infection and a clinical specificity of 99.9% according to the manufacturer.recomLine SARS-CoV-2-IgG/ CE-IVD allows the differentiation of antibodies against seasonal coronaviruses  as well as the following proteins of SARS-CoV-2: NP SARS-2, RBD-SARS-2 and S1-SARS-2. Diagnostic sensitivity is stated as 85.7% for less than 12 days after infection, 95.2% for 12 to 23 days after infection and 100% if the infection occurred more than 23 days after diagnostics. This test has a very high diagnostic specificity according to the manufacturer with 97.9% for potentially cross-reactive samples and 99.7% in blood-donors.The Mikrogen® Parents and teachers or caregivers were only tested with the rapid lateral-flow test. If their result was positive for IgG antibodies, the capillary blood of these participants was again examined via dried blood spots for SARS-CoV-2 antibodies using the Euroimmun® ELISA (see above).Descriptive analysis included the participation rate of schools and preschools. Medians and interquartile ranges (IQRs) were calculated. The proportion of positive tests (IgG) was determined. IgG antibodies measured by Euroimmun® ELISA were assessed in RE/ml, and then converted to Binding Antibody Units (BAU)/ml. BAU/mlSARS-CoV-2-IgG antibodies were presented stratified by positive/negative result for time-point comparisons and questionnaire data. When measuring IgG antibodies in the Euroimmun® ELISA, the lowest level of quantification is 2.4 BAU/ml. The highest measurable value was given as >288 BAU/ml. This value was set to 288.1 BAU/ml for all calculations. The percentage of not available values (missings/ NAs) is reported. NA values are excluded from the calculation of all statistical measures . For subgroup analyses, the size of the group is always reported in absolute numbers. Groups were first tested globally for differences using the Friedman test in the case of more than two groups. Pearson’s Chi-squared test, Fisher’s exact test, or Wilcoxon rank sum test were used when computing post-hoc tests or when testing two groups against each other. A statistically significant difference was assumed at a level of p<0.05. When making group-comparisons in symptomatic versus asymptomatic children, we divided symptoms into two groups: Symptoms since the start of the pandemic  and symptoms four weeks before baseline examination . Symptoms were grouped by time of onset (symptoms since the start of the pandemic and symptoms since 4 weeks before baseline examination). If the child ticked one symptom in either group of the groups, the corresponding variable was set as “Yes”. The R Studio ©, version 4.1.3, was used for statistical analyses.A total of 365 children and 331 parents as well as 90 educators or teachers were included in the study. Almost 55% of the children were male and most of the children attended secondary school (40.5%). Of the participating children, 56.0% were in the age group 3–10 years, and 44.0% in the age-group 11–16 years. Mostly mothers (71.9%) and female educators and teachers (86.7%) took part in the study. The median age of the parents was 43.0 years  and that of the teachers and educators 44.5 years  . The medrecomLine blot  tested positive for IgG antibodies at the baseline visit . Only 12When looking at IgG-positive children at baseline, children with symptoms had higher median IgG levels (BAU/ml), but the difference between symptomatic and asymptomatic children was not significant Figs  and 6. TWhen stratifying the children by IgG antibody status (positive/negative), we found that significantly more IgG-positive children also had an IgG-positive parent (p<0.001). In addition, significantly more IgG-positive children also had a positive PCR test (p<0.001), and/or one parent had a positive PCR test (p<0.001) before study entry. All IgG-positive children had contact with a person with COVID-19 at some point (p<0.001). Looking at the hygiene behavior of the children, significantly more IgG-negative children are in the group of those who wash or disinfect their hands more frequently since the start of the pandemic (p = 0.037). There was no significant difference in IgG antibody outcomes between facility types . NeverthrecomLine blot and those in which an infection was identified via the detection of IgG to SP1 in the Roche® ECLIA. However, one sample with a weak IgG detection in both Roche® ECLIA and recomLine blot was below the cut-off in the Euroimmun® ELISA using dried blood spot.The Of the 16 children who tested positive for antibodies to SP1 during the entire study period, only nine reported having a previous SARS-CoV-2 infection . In a reIn the present study, SARS-CoV-2 seroprevalence of 3.6% (n = 13) was measured at baseline using Euroimmun® dried blood spots. The adults in our cohort had a comparable or slightly higher prevalence as observed in the children: We measured an IgG seroprevalence of 4.3% (n = 13) and 3.7% (n = 3) for parents and educators/teachers at baseline, respectively.Twelve of the IgG-positive children could be confirmed by the Roche® Anti-SARS-CoV-2-ECLIA using venous blood samples . However, at baseline only six (50.0%) of the 12 children with confirmed SARS-CoV-2 infections reported having been ill with the disease–suggesting an underdiagnosis of SARS-CoV-2 infections in children during the Augsburg-Plus study period. Our finding of a 50.0% underdiagnosis of SARS-CoV-2 infection in children is in accordance with the results of two further investigations ,11, althTo date, few studies have measured SARS-CoV-2-specific IgG antibodies in children in order to determine whether a child has previously experienced a symptomatic or asymptomatic infection with SARS-CoV-2 –14,18–24It may be assumed that immunity following a SARS-CoV-2 infection lasts approximately as long as with SARS-CoV, which is up to 24 months after infection . The forSARS-CoV-2 seroprevalence in our study was approximately 3.5%, which is higher than what was measured in a Swiss (regarding the group of children aged 5–9 years ), and twRostami et al. conducted a systematic review of seroprevalence in the general population with studies published up to August 2020 worldwide. For children/adolescents younger than 19 years, a seroprevalence of 2.3% (95% CI 1.0–3.6) was calculated when reviewing all data sets .Hommes et al. conducted a cohort study in Berlin, which is comparable to our study. The data collection took place in June 2020. In this study, children and teachers from various primary and secondary schools were also recruited. They identified only one positive PCR test over the course of the study period in a 16-year-old girl among a total sample of 382 tested participants . HoweverIn our study, six of a total of twelve confirmed seropositive children (50.0%) at baseline did not know that they had undergone SARS-CoV-2 infection. The finding of an underdiagnosis of SARS-CoV-2 infection in children is in accordance with two other investigations ,11 althoIgG antibodies decreased significantly over time but persisted in 100% over a period of 4 months in our study. This confirms the result of Renk et al., who still measured IgG antibodies in 96.2% of their cohort including 548 children aged 6–13 years 11–12 months after baseline examination .Altogether, three seroconversions occurred during the 4-months follow-up, two of which occurred in children attending secondary school. In the parents of the children, we detected one case of seroconversion, and none in educators/teachers. These cases must be seen in the context of the incidence of infection in the population and the public measures taken to contain the pandemic. The comparatively few to no seroconversions among adults in the cohort may be due to the high vaccination-rate. Within 4 months after baseline, 66.1% of the parents, and 94.1% of educators/teachers had been vaccinated at least once. Fischer et al. measured an IgG seroprevalence of 0.9% within a cohort of more than 3000 adults from three German states, who donated blood between March 2020 and June 2020 , which iWe demonstrated the capability of antibody testing out of capillary blood in children and used two different systems, including an ELISA out of dried blood spots (Euroimmun®) by comparing these results to antibodies from venous blood samples. Furthermore, we detected that two of the children who screened positive by NADAL® rapid test were actually infected with the seasonal human coronavirus NL63 instead, and one additional child with a soft reaction in IgG had no proof of SARS-CoV-2-antibodies in any further assay.The results of the present study could be biased by the fact that participation in the study was based on voluntary enrolment by parents. However, we tried to counteract this by restricting participation only to children from the participating facilities. Within the facilities, the facility managers repeatedly called for participation. Unfortunately, we could recruit comparatively fewer children from preschools than from other facilities for the study. We tried to compensate for this by working with more preschools. As the vaccination campaign for adults started while our study was conducted, we were somewhat limited in terms of determining seroprevalence. In addition, children who may have developed only a cellular immune response (T-cells) remained undetected in our study. Furthermore, the results always have to be considered in the context of the different phases of the pandemic, the differently implemented measures of outbreak control, the included study participants and the serologic test used for the antibody determination. The assays we used perform differently depending on how many days after symptom onset the test is done . In addiOne strength of the study is first and foremost that we used two different test systems for the screening for antibodies and were able to obtain a more accurate picture of actual SARS-CoV-2 cases by recomLine blotting than was initially indicated by ELISA or even rapid testing. This allowed us to sort out those antibody reactions that were due to cross-reactivity to seasonal coronaviruses .Multiple study time points also allowed us to capture seroconversions. Our study took place during the third wave of the pandemic, i.e. at a more recent point in time than previous studies on seroprevalence. Thus, our results reflect current conditions on seroprevalence in 3-16-year-old children and complement previous studies on this topic.The combination of two antibody test systems, both out of capillary blood, proved to be a reasonable way to test children with minimal invasiveness combined with an acceptable sensitivity. Results of our study showed a seroprevalence of SARS-CoV-2 antibodies of 3.6% in children aged 3–16 years. However, it seems that SARS-CoV-2 infections in children were underdiagnosed during the time of data collection of the Augsburg Plus data. More data on SARS-CoV-2 seroprevalence in children will be useful, especially longitudinal data, which map the IgG antibody course over a longer time period. In Germany, 20.0% children of the 5–11 year old children of age are currently vaccinated . For theS1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Molecular mechanisms underlying the production of livestock are of great interest at all times. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide; it takes part in the regulation of various physiological processes, such as feeding, reproduction, thermoregulation and brain development. This study was conducted to investigate the effect of high plasma PACAP levels and low plasma PACAP levels on the protein composition of blood plasma. Our finding is that plasma PACAP level is associated with the abundance of 10 plasma proteins. The functions of the differentially expressed proteins indicate that the PACAP level of plasma is related with the lipid metabolism and immune status of cattle.p < 0.05) in the expression levels between groups. Ten spots showed a higher intensity in the high-PACAP-concentration group, while six spots were more abundant in the low-PACAP-concentration group. The functions of the differentially expressed proteins indicate that the PACAP level of plasma is related to the lipid metabolism and immune status of cattle.Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide; it takes part in the regulation of various physiological processes, such as feeding, reproduction, catecholamine synthesis, thermoregulation, motor activity, brain development and neuronal survival. Since PACAP plays important regulatory roles, we hypothesized that the level of PACAP in blood is associated with expression of other proteins, which are involved in different metabolic pathways. The objective of the present study was to compare plasma protein profiles of cows with high and low plasma PACAP levels. Differential proteome analyses were performed by two-dimensional gel electrophoresis (2D-PAGE) followed by tryptic digestion and protein identification by liquid chromatography–mass spectrometry (LC-MS). A total of 210 protein spots were detected, and 16 protein spots showed statistically significant differences ( Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide and a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon peptide family . NeuropePACAP is found in two bioactive forms, PACAP27 and PACAP38, of which PACAP38 is the major form in mammalian tissues . Both peOnly a few papers have been published so far on the presence and functions of pituitary adenylate cyclase-activating polypeptide in livestock species ,9,10,11.The objective of the present study was to compare plasma protein profiles of cows with high and low plasma PACAP levels. A total of 74 Holstein cows were included in the study for blood sampling, and 10 of them were used for further analysis. All cows were in their second or third lactation, and pregnant cows were excluded from the study. Cows were housed at a conventional dairy farm and fed with corn silage supplemented with concentrate. Blood sample was collected from the tail vein, placed in a sterile 9 mL blood tube containing 150 µL of protease inhibitor aprotinin  and cooled and centrifuged at 2000 rpm for 10 min at 4 °C. Plasma was removed from each sample, divided into aliquots and immediately frozen at −80 °C until further analysis. Study was conducted according to the guidelines imposed by the European Union Directive 2010/63/EU on the protection of animals used for scientific purposes.Bovine PACAP enzyme-linked immunosorbent assay kit  was used to measure the plasma PACAP concentration. Assay was performed according to the manufacturer’s instructions. Plasma samples were diluted 100 times, and all samples and standards were measured in duplicate. Absorbance data were collected with a SPECTROstar Nano  microplate reader set to 450 nm, and then raw ODs were corrected by subtraction of absorbance measured at 540 nm. Sample concentrations were interpolated from a standard curve created by four-parameter logistic curve fit using MARS data analysis software 3.10 . PACAP level of 74 cows was determined, and 5 cows with plasma PACAP level below 100 pg/mL were grouped into the low-PACAP-level group  and 5 cows above 500 pg/mL were allocated to the high-PACAP-level group .w/v) CHAPS, 40 mM DTT, 0.2% (v/v) Bio-Lyte 4/6 and 6/8 ampholyte at a ratio of 1:2). The protein concentration was determined using RC DC protein assay kit  with bovine serum albumin (BSA) as standard. The protein concentration of the samples was in the range of 4.2–4.8 mg/mL.Plasma samples, containing up to 500 µg of protein in a final volume of 100 µL, were precipitated following the protocol provided with ReadyPrep 2-D Cleanup Kit . The procedure works by quantitatively precipitating and concentrating proteins in a sample while leaving behind and washing away substances such as ionic detergents, nucleic acids, lipids and salts, all of which are known to interfere with IEF. After precipitation, the protein pellets were washed and then resuspended in rehydration buffer  CHAPS, 40 mM DTT, 0.2% (v/v) Bio-Lyte 4/6 and 6/8 ampholyte at a ratio 1:2, 0.002% (w/v) bromophenol blue) for 16 h at room temperature. A total of 200 µg of protein was loaded, and the isoelectric focusing was conducted in Protean IEF Cell . Low voltage (250 V) was applied for 20 min, and then the voltage was gradually increased to 4000 V over 2.5 h and maintained at that level until a total of 20,000 Vh was reached. The current limit was adjusted to 50 mA per strip, and the run was carried out at 20 °C. Focused IPG strips were equilibrated for 10 min in 6 M urea, 20% (v/v) glycerol, 2% (w/v) SDS, 50 mM Tris pH 8.8 and 2% (w/v) DTT, and then for an additional 10 min in the same buffer with the exception that DTT was replaced by 2.5% (w/v) iodoacetamide. After equilibration, proteins were separated in the second dimension using OmniPAGE Mini  vertical electrophoresis system. Second dimension was performed on 100 × 100 mm, 13% polyacrylamide gels (37.5:1 acrylamide:bis-acrylamide ratio). The gels were run by applying 80 V in the first 10 min and then 180 V until the bromophenol blue dye marker reached the end of the gels. A cooling system provided a constant running temperature of 20 °C. Polyacrylamide gels were stained with colloidal coomassie G-250  overnight [For the first dimension (isoelectric focusing) of two-dimensional gel electrophoresis, 7 cm immobilized pH gradient strips  were rehydrated by passive rehydration using samples dissolved in 125 µL of rehydration buffer . Polyacrylamide gel images were scanned using PharosFX Plus  fluorescent system, and images were analysed using Delta2D software . For gel analysis, all gel images within a group were warped to the first gel image. Spots were automatically detected, and images were checked by eye for undetected or incorrectly detected spots. A reference gel was created by fusing all images using union fusion. Every spot on each gel was quantified and normalized according to the total intensity of all spots on each gel. Student’s p < 0.05) intensity difference between the low- and high-PACAP-concentration groups using GraphPad Prism 9.3.1. The optimal number of PCs was found to be 2. The loading plot of PC1 and PC2 was examined to reveal the spot differences between the low- and high-PACAP-concentration groups.Principal component analysis (PCA) was performed on spot volumes with statistically significant  acetonitrile in 25 mM ammonium bicarbonate solution. Trypsin digestion was carried out using stabilized MS grade bovine trypsin (12.5 ng/µL) at 37 °C overnight. Peptides were extracted with 5% (v/v) formic acid, and washed twice with 60% (v/v) acetonitrile in 1% formic acid. The digested peptides were concentrated with speed-vac and redissolved in 10 µL of 1% formic acid (v/v). Protein identification was carried out in the University of Debrecen Proteomics Core Facility. The spots were manually cut out from the gel in a laminar flow cabinet to prevent keratin contamination and destained by washing with 50%  mass spectrometer coupled to an Easy nLC II nanoHPLC . The peptides were purified on a Zorbax 300SB-C18 precolumn  and separated on a reverse-phase Zorbax 300SB-C18 analytical column  using a 90 min water–acetonitrile gradient and 300 nL/min flow rate. Information-dependent acquisition was performed, and the two most intensive ions were used for MS/MS. During analyses, the spray voltage was 2800 V, the nebulizing gas was 50 psi, the curtain gas was 10 psi, the source temperature was 70 °C, and the declustering potential was 50 V.The acquired LC-MS/MS spectra were used for protein identification; the ProteinPilot 4.5  search engine, the Uniprot/SwissProt database with no species restriction, trypsin as the modifying enzyme and the biological modification table included in the ProteinPilot 4.5 were managed. For protein identification, a minimum of two peptides with more than 95% confidence was required.https://string-db.org/ accessed on 6 June 2021), and a maximum of 50 first-shell interactors of the query proteins were used under maximum stringency (0.9). The networks and the enriched gene ontology (GO) functions were examined. Two networks were generated, one for each studied group. The bovine immunoglobulins were not found in the String database, so they were omitted from the analyses.Proteins identified in spots with statistically significant changes between the groups, plus PACAP, were included in a network analysis. For network generation, the String database was used (p < 0.05) in the expression levels between the high- and low-PACAP-concentration groups  intensity differences between the low- and high-PACAP-concentration groups. PCA is a statistical method for determination of the key variables in a multidimensional dataset that explains the differences in the observations. Individual principal component ‘loadings’ represent the contribution of individual protein spot volumes to the variation in the data. This enables the demonstration of the relative contribution of these proteins to proteome differences present between high-PACAP and low-PACAP-plasma-level cows, as well as the identification of clusters of proteins that behave similarly. Protein spots with higher intensities in one of the cow groups based on their PACAP plasma levels appear in the same cluster of the PCA plot. For example, spots 184, 260, 280, 285, 287 and 310 all showed higher volumes in the low-PACAP-level group, and loadings are close to each other on the PCA plot  was performed on spot volumes with statistically significant  and 1.5-fold (p = 0.002) higher expression in the high-PACAP group, respectively. Bf is a serine protease, primarily synthesized by the liver, which activates the alternative pathway of the complement system [Some of the proteins identified in the selected spots have a role in the immune response. Complement factor B (Bf) was identified in spot 63 and 65 showing 1.4-  and, in spot 150, 1.2-fold (p = 0.004) higher expression. Contrary to Ig heavy chains, Ig gamma-2 chain C region protein in spot 260 and IGL@protein in spot 280 showed higher expression (p < 0.000) in the low-PACAP-concentration group. Immunoglobulins are produced by B cells to identify and neutralize pathogens. Up-regulation of Ig heavy chain was observed in the high-PACAP-concentration group at three different positions on the polyacrylamide gels, showing 1.3-fold higher expression in spots 145 and 152 (p < 0.000) in the high-PACAP-concentration group. In spot 310, C4b-binding protein alpha chain was identified, which is mainly produced in the liver. C4b-binding glycoprotein (C4BP) is the most important soluble inhibitor of the classical and lectin pathways of the complement system . The comp = 0.019 and 0.003) in the low-PACAP-concentration group. ApoA1 plays a role in cellular cholesterol efflux, and it has antioxidant and anti-inflammatory effects [p = 0.003) intensity in the low-PACAP-concentration group. Apolipoprotein A-IV (apoA4) acts primarily in intestinal lipid absorption; fat absorption increases its synthesis and secretion by the small intestine [The other class of identified proteins belong to lipid-binding apolipoproteins. They regulate lipoprotein metabolism and transfer and redistribution of lipids among tissues . Apolipo effects ,27. Onts effects . Apolipontestine . ApoA4 mntestine . Wang anntestine  also demntestine .p = 0.008, p = 0.002, p = 0.002, p = 0.003) in the high-PACAP-concentration group. Serotransferrin is an abundant single-chain glycoprotein in plasma which is synthetized in the liver. Its main function is to bind and transport iron, but it plays an important role in many other physiological processes, such as: stimulation of cell proliferation [Besides the proteins with a role in immune functions or lipid metabolism, proteins with transport functions have also been identified. Four protein spots  contained serotransferrin. These spots were located at different pI positions, which might be the consequence of post-translational modifications influencing the charge and size of the protein. The expression of each spot was higher  expression in the high-PACAP-concentration group. The main function of the retinol-binding protein 4 (RBP4) is transporting retinol from the liver to the peripheral tissues. The vitamin A deficiency blocks its secretion post-translationally [Retinol-binding protein 4 (in spot 299) had 1.6-fold higher (tionally . RBP4 wationally , inducintionally . The obetionally .Our results indicate that PACAP concentration is related to the lipid metabolism and immune status of cattle. Previous studies have demonstrated that the pituitary adenylate cyclase-activating polypeptide plays an important role in lipolysis and lipogenesis, as well . In rat The existence of cross-talk between the neuroendocrine and immune system is established through mediators such as PACAP which exhibit potent immunoregulatory properties. PACAP has the ability to modulate the innate and adaptive inflammatory responses at multiple sites . In the The higher level of immune-system- and defense-related proteins in the high-PACAP-concentration group indicates the involvement of the PACAP in the regulation of the immune functions.At the same time, in the low-PACAP-concentration group, the spots containing proteins with a role mainly in lipid and lipoprotein metabolism showed higher intensity. It is very likely that a PACAP has a complex regulatory mechanism in the adaptive and innate immune system, as spots containing proteins with a role in the immune system showed higher intensity in both groups. Based on the generated protein–protein interaction networks, it is very likely that the proteome-level changes observed in this study are not the result of the direct effect of PACAP on these proteins. Considering that PACAP is a hormone acting on transmembrane receptors, its functions point beyond direct protein–protein interactions and can have broader effects modulated by the initiated signal transduction pathways.Our study provides useful information on the proteome-wide changes in cow plasma elicited by PACAP, and in order to deepen our knowledge, further studies are needed, such as the comparative analysis on PACAP-injected vs. noninjected cows to evaluate the changes in proteome.The results of two-dimensional gel electrophoresis followed by a mass spectrometry proteomic approach obtained from the present study indicate that the concentration of pituitary adenylate cyclase-activating polypeptide in the blood plasma is closely related to the lipid metabolism and immune status of dairy cows.


Staphylococcus aureus osteomyelitis remains a very challenging condition; recent clinical studies have shown infection control rates following surgery/antibiotics to be ~60%. Additionally, prior efforts to produce an effective S. aureus vaccine have failed, in part due to lack of knowledge of protective immunity. Previously, we demonstrated that anti-glucosaminidase (Gmd) antibodies are protective in animal models but found that only 6.7% of culture-confirmed S. aureus osteomyelitis patients in the AO Clinical Priority Program (AO-CPP) Registry had basal serum levels (>10 ng/ml) of anti-Gmd at the time of surgery (baseline). We identified a small subset of patients with high levels of anti-Gmd antibodies and adverse outcomes following surgery, not explained by Ig class switching to non-functional isotypes. Here, we aimed to test the hypothesis that clinical cure following surgery is associated with anti-Gmd neutralizing antibodies in serum. Therefore, we first optimized an in vitro assay that quantifies recombinant Gmd lysis of the M. luteus cell wall and used it to demonstrate the 50% neutralizing concentration (NC50) of a humanized anti-Gmd mAb (TPH-101) to be ~15.6 μg/ml. We also demonstrated that human serum deficient in anti-Gmd antibodies can be complemented by TPH-101 to achieve the same dose-dependent Gmd neutralizing activity as purified TPH-101. Finally, we assessed the anti-Gmd physical titer and neutralizing activity in sera from 11 patients in the AO-CPP Registry, who were characterized into four groups post-hoc. Group 1 patients (n=3) had high anti-Gmd physical and neutralizing titers at baseline that decreased with clinical cure of the infection over time. Group 2 patients (n=3) had undetectable anti-Gmd antibodies throughout the study and adverse outcomes. Group 3 (n=3) had high titers +/− neutralizing anti-Gmd at baseline with adverse outcomes. Group 4 (n=2) had low titers of non-neutralizing anti-Gmd at baseline with delayed high titers and adverse outcomes. Collectively, these findings demonstrate that both neutralizing and non-neutralizing anti-Gmd antibodies exist in S. aureus osteomyelitis patients and that screening for these antibodies could have a value for identifying patients in need of passive immunization prior to surgery. Future prospective studies to test the prognostic value of anti-Gmd antibodies to assess the potential of passive immunization with TPH-101 are warranted. Staphylococcus aureus (S. aureus (MRSA) in some regions  and often requires multiple surgeries  gen evasion , (3) int evasion , and (4) evasion . As a reurgeries .S. aureus immunizations have been evaluated in Food and Drug Administration (FDA) registration trials, and all failed to demonstrate efficacy (S. aureus enzymes and (2) immunomodulatory activity to stimulate the host response and bacterial clearance (S. aureus autolysin (Atl) as our lead target for passive immunization  with high affinity and 1:1 stochiometric neutralizing activity  had basal levels of anti-Gmd antibodies (>10 ng/ml) in their serum at the time of surgery (baseline). Of these patients, 194 (65.3%) completed the 1-year follow-up and were divided into groups based on their anti-Gmd antibody level at baseline, namely, low , intermediate , and high , and the infection control rates were 40.7%, 50.0%, and 66.7%, respectively. The incidence of adverse outcomes in these groups was 33.3%, 16.4%, and 11.1%, respectively. While high anti-Gmd titers were not the only deciding factor in infection control, as 21 out of 194 patients (10.8%) had low titers and achieved a favorable outcome at 1-year post-surgery, by assessing anti-Gmd level as a continuous variable, we found that for every 10-fold increase in concentration, there was a 60% reduction in adverse event risk (p=0.04). Furthermore, patients with low anti-Gmd titer demonstrated a highly significant 2.7-fold increased risk in adverse outcomes (p=0.008). However, a few of these patients had high titers of anti-Gmd antibodies at baseline and had adverse outcomes following surgery, which was not due to IgG4 class switching to non-functional immunoglobulin -approved protocols . Specific serum samples from the AO Trauma Clinical Priority Program (CPP) Bone Infection Registry were selected for study based on their known anti-Gmd physical titer and the patient’s clinical outcome .via protein-A affinity chromatography  was used as a substrate for recombinant His-Gmd at final concentration of 0.075% (750 μg/ml) in phosphate-buffered saline (PBS) as we previously described  from OD600 of M. luteus treated with Gmd neutralized by various concentrations of TPH-101 antibody and dividing the product by the OD600 obtained from subtracting OD600 of M. luteus treated with Gmd (10 μg/ml) from OD600 of M. luteus (bacteria only). IBT produced antibody c21D10  (6.643 mg/ml) was used as negative isotype control. The neutralizing concentration (NC50) value was determined using Sigmoidal 4PL, where X is concentration, and the least squares fit was used to quantify the 50% (NC50) of TPH-101 in the 30- and 60-min incubation.Purified TPH-101 (1 mg/ml) was serially diluted in PBS, and equal volume of 40 μg/ml Gmd was added in a 96-well plate and incubated at 37°C for 15 min. After incubation, equal volume of 0.15% heat-killed M. luteus treated with 1% Triton X-100 was added. OD600 was measured after incubating at 37°C for 30 and 60 min, and percentage of neutralization was calculated as above.Human serum samples were heat inactivated for 30 min at 56°C. Equal volume of heat-inactivated human serum samples (neat) and Gmd (40 μg/ml) was incubated at 37°C for 15 min. After incubation, equal volume of 0.15% 600 was measured after 30 and 60 min. TPH-101 was used as positive control, c21D10 was used as negative control, and non-spiked serum was used as non-neutralizing control.Human serum sample without physical titers against Gmd (ELISA) from the AO Clinical Priority Program (AO-CPP) cohorts were diluted 1:20, and 1 mg/ml of TPH-101 was added and incubated at 37°C with equal volume of Gmd (40 μg/ml) for 15 min. After incubation, the substrate was added, and ODM. luteus cell wall digestion assay by solubilizing the substrate in varying concentrations of a non-ionic detergent (Triton X-100). M. luteus digestion.Prior to assessing the Gmd autolysis-neutralizing activity of the patient sera , we aimed to optimize the 50) of Gmd enzyme activity in the M. luteus cell wall lysis assay, we performed a dose–response study using the optimized in vitro conditions described in 50 of ~15.5 μg/ml (M. luteus and assessed cytolysis via colony-forming unit (CFU) assay, which demonstrated a similar NC50 of ~12.5 μg/ml (−1 to 10−6), and 100 μl was plated on tryptic soy agar (TSA) plates, and colonies were counted by incubating at 37°C for 48 h.To determine the concentration of purified TPH-101 required to neutralize 50%  had high physical titers of anti-Gmd antibodies at baseline (>10 ng/ml), (2) never had detectable anti-Gmd titers throughout their treatment, or (3) developed high titers of anti-Gmd at some point during their treatment. The results are presented with the clinical outcomes in S. aureus osteomyelitis patients and that screening for the types of antibodies could have value for identifying patients in need of passive immunization prior to surgery.Although we have previously described the association of anti-Gmd antibody physical titers with clinical outcome of the patients in the AOTrauma CPP Bone Infection Registry , the GmdWe were also interested to know if anti-Gmd antibody physical titers correlate with Gmd neutralizing activity in the patient sera. Thus, we performed a linear regression analysis on the five sera samples that contained Gmd neutralizing activity, and our negative findings are presented in S. aureus would be transformative for orthopedic surgery and many other infections caused by this pathogen. Here, we have focused on the hypothesis that an ideal mAb would act both directly via antimicrobial effects through inhibition of a critical S. aureus target and have immunomodulatory activity to enhance the host response and bacterial clearance. From non-biased antigen discovery, in vitro, animal model, and clinical research, we identified Gmd as a validated target for immunotherapy  in their serum at the time of surgery, and that for every 10-fold increase in anti-Gmd antibody concentration in sera, there is a 60% reduction in adverse event risk  or develop them too late in the disease process (Group 4) indirectly supports our hypothesis of anti-Gmd mAb therapy. It was also interesting to see that some patients who develop high titers of non-neutralizing antibodies also succumb to serious adverse events from S. aureus infection (Group 3) and that anti-Gmd physical titer does not correlate with Gmd neutralizing activity  neutralizing anti-Gmd antibodies, (2) non-neutralizing antibodies, and (3) undetectable anti-Gmd antibodies at the time of their surgery are needed to validate the association of neutralizing antibodies with clinical outcome. It is also important to note that some clinical outcomes do not have a straightforward interpretation. For example, patient 8 had high titers of neutralizing anti-Gmd antibodies at baseline and had a knee fusion that we scored as an “Adverse” outcome based on our prospective criterion. However, this successful infection control, potentially aided by the patient’s anti-Gmd antibodies, may have been the best possible outcome based on the patient’s global health (76 years old with Class III obesity) and the damaged bone and soft tissue at the time of surgery. Finally, while the M. luteus cell wall digestion assay proved very useful for these research studies, we do not suggest that it can be translated into a clinical diagnostic due to the technical demands of the assay. Thus, efforts to develop a lateral flow assay to assess anti-Gmd as a biomarker are warranted.As a small clinical pilot, there are several major limitations that need to be noted. In addition to the minimal numbers of patients studied, which are too small to make formal conclusions other than both neutralizing and non-neutralizing anti-Gmd antibodies existing in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Virginia Commonwealth University HRPP. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by University of Rochester.All authors were directly involved in designing the experiments, data analysis, and drafting the manuscript. SS, GM, TK, and JO performed experiments. SK is the principal investigator of the IRB-approved clinical research. All authors contributed to the article and approved the submitted version.This work was supported by research grants from the National Institutes of Health  and the AOTrauma Clinical Priority Program.SS, RA, TK, and MA are paid employees of Integrated Biotherapeutics Inc. RP and ES are paid consultants of Integrated Biotherapeutics Inc. and have stock in Telephus, LLC.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Flavobacterium sp. No. 92. Although most cyclomaltodextrinases have been classified in the subfamily GH13_20. This one has not been assigned any GH13 subfamily as yet. It possesses a non-specified immunoglobulin-like domain at its N-terminus mimicking a starch-binding domain (SBD) and the segment MPDLN in its fifth conserved sequence region (CSR) typical, however, for the subfamily GH13_36. The searches through sequence databases resulted in collecting a group of 108 homologs forming a convincing cluster in the evolutionary tree, well separated from all remaining GH13 subfamilies. The members of the newly proposed subfamily share a few exclusive sequence features, such as the “aromatic” end of the CSR-II consisting of two well-conserved tyrosines with either glycine, serine, or proline in the middle or a glutamic acid succeeding the catalytic proton donor in the CSR-III. Concerning the domain N of the representative cyclomaltodextrinase, docking trials with α-, β- and γ-cyclodextrins have indicated it may represent a new type of SBD. This new GH13 subfamily has been assigned the number GH13_46.In the CAZy database, the α-amylase family GH13 has already been divided into 45 subfamilies, with additional subfamilies still emerging. The presented in silico study was undertaken in an effort to propose a novel GH13 subfamily represented by the experimentally characterized cyclomaltodxtrinase from  Within the CAZy database sequence-based classification of glycoside hydrolases (GHs) , the fam8-barrel (TIM-barrel) as a catalytic domain employing the retaining reaction mechanism; (ii) the catalytic machinery consists of a catalytic nucleophile (aspartic acid), a proton donor (glutamic acid) and a transition-state stabilizer (aspartic acid) at the strands β4, β5 and β7, respectively; and (iii) sequences share from 4 up to 7 typical conserved sequence regions (CSRs). The canonical domain organization in the family GH13 consists of three domains: A , B (it protrudes out of the barrel in the place of the loop 3 connecting the strand β3 with the helix α3), and C , although various mainly starch-binding domains (SBDs) classified as a different carbohydrate-binding module (CBM) families being also found [The family GH13 can briefly be characterized by a few basic criteria as follows ,11,12,13so found .At the higher level of the CAZy hierarchy, the family GH13 has been grouped with related families GH70 and GH77 into the clan GH-H , whereasFlavobacterium sp. No. 92, whose three-dimensional structure was published already in 2003 [Bacteroides thetaiotaomicron [Zunongwangia profunda [Massilia timonae [Flavobacterium sp. No. 92 cyclomaltodextrinase, all the particular features of interest mentioned above.The present study has been undertaken in an effort to emphasize the relevancy of creating a novel GH13 subfamily around the cyclomaltodextrinase from  in 2003 . The enz in 2003  and subs in 2003 ,27,28. D in 2003 , this cy in 2003 . The rea in 2003 ,32,33,34 in 2003 ,23. Note in 2003 . Finallyaomicron , an α-amprofunda  and a cy timonae , which sFlavobacterium sp. No. 92 is an attractive subject worth deep in silico studies that should be helpful in elucidating its position within the entire α-amylase family GH13. Therefore, the aim of the present study has been to deliver the comprehensive results from such a bioinformatics analysis convincing enough in order to define a novel GH13 subfamily, the subfamily GH13_46, represented just by this unique cyclomaltodextrinase.All these attributes make thus the cyclomaltodextrinase from Flavobacterium sp. No. 92 [Bacteroides thetaiotaomicron [Zunongwangia profunda [Massilia timonae [http://www.cazy.org/, accessed on 29 October 2022), and they all share two specific features: (i) the intermediary character of the sequence in the fifth CSR—MPDxN [https://blast.ncbi.nlm.nih.gov/, accessed on 1 October 2020), using the amino acid sequence of the Flavobacterium cyclomaltodextrinase (UniProt Accession No.: Q8KKG0) as a query. In total, three searches with the same cyclomaltodextrinase query sequence were performed, i.e., separately limiting the searched databases to kingdoms of Bacteria, Archaea and Eucarya. With regard to sources of sequences caught by BLAST, one non-redundant amino acid sequence was selected to represent each species and/or bacterial strain. Furthermore, the simultaneous presence of three sequence-structural features was considered as basic criterium for sequence selection: (i) the N-terminal module homologous to that present in the cyclomaltodextrinase from Flavobacterium sp. No. 92 [The cyclomaltodextrinase from . No. 92 , neopullaomicron , α-amylaprofunda , and cyc timonae  were sel timonae ; http://SR—MPDxN ; and  up. No. 92  with a s. No. 92 ; and  six representatives of the most recently established subfamily GH13_45—three members grouped around the α-amylase BaqA from Bacillus aquimaris [Bacillus megaterium [In order to perform a convincing comparison covering the entire α-amylase family GH13, the selected set of the sequences forming the potential novel GH13 subfamily has been further completed by 145 sequences as follows : (i) thrquimaris ,20 and tgaterium . Sequenchttps://www.uniprot.org/, accessed on 26 November 2022) and/or GenBank  databases.All studied sequences were retrieved from the UniProt . The first three alignments were executed on 108 Flavobacterium cyclomaltodextrinase-like sequences (that should define the new GH13 subfamily), whereas the fourth alignment was done with the full set of 253 sequences . While the former alignments (108 sequences) were based on: (i) N-terminal modules; (ii) the GH13 canonical part of the enzymes ; and (iii) the full-length enzymes; the latter alignment (253 sequences) was based on the substantial part of the catalytic domain spanning the sequence segment from beginning of the CSR-VI (strand β2) to the end of the CSRV-VII (strand β8) including the domain B. Information about the domain boundaries and all CSRs of individual sequences was obtained from the literature describing the sequences of biochemically characterized members of the individual subfamilies and previous in silico studies [Four different sequence alignments were performed using the program Clustal-Omega . For the trees of the newly proposed GH13 subfamily, the branch swap filter parameter has been set to “very strong,” and all other specifications were used in a default mode. Finally, all four calculated tree files were displayed with the program iTOL .Four evolutionary trees were calculated for the four above-mentioned sequence alignments. All were calculated as maximum-likelihood trees  using the WAG substitution model  and the am iTOL .The sequence logo of seven well-established CSRs for all 108 sequences of the potentially novel GH13 subfamily represented by the cyclomaltodextrinase from  server  for (i) cyclomaltodextrinase from Flavobacterium sp. No. 92 .Three-dimensional structures were retrieved from the Protein Data Bank .The full-length tertiary structure of the cyclomaltodextrinase from . No. 92  was used. No. 92  was comp. No. 92  as well x orenii . All terx orenii ; http://Flavobacterium sp. No. 92 may eventually function as an SBD. The docking trials using the program AutoDock and MGL Tools v1.5.6 [https://pubchem.ncbi.nlm.nih.gov/, accessed on 30 September 2021) and converted into PDB coordinates by the SMILES program . The resulting complexes of individual structures with bound cyclodextrins were displayed using the UCSF Chimera program [In order to inspect whether or not the N-terminal domain of the cyclomaltodextrinase from s v1.5.6  were pers v1.5.6 ; PDB cods v1.5.6 ; https:/program  neaomicron ; (iii) αprofunda ; and , which is a microscopic single-celled flagellated protozoan parasite [Bacteroides thetaiotaomicron [Euryarchaeota archaeon (UniProt: A0A2E0K4E8), it has been placed in the largest cluster where the Flavobacterium sp. No. 92 cyclomaltodextrinase [In order to follow the evolutionary relationships within the new GH13 subfamily, the evolutionary tree based on the alignment of complete sequences was calculated . Since oparasite , has beeaomicron . With rextrinase  is foundFor comparison, two further corresponding evolutionary trees were constructed: (i) based on the alignment of family GH13 canonical domains A, B, and C, i.e., excluding the domain N ; and ii. It is wTritrichomonas foetus , which can provide possible stacking interactions  the N-terminal domain not compatible with the SBD of the family CBM34  that is typically from the CAZy subfamily GH13_20  and is nly CBM34  present ly CBM34 , for whiaomicron , the α-aprofunda  and the  timonae  but alsoWith regard to relationships within the new subfamily, three evolutionary trees have been constructed based on the alignment of (i) complete sequences ; (ii) seFlavobacterium sp. No. 92 may strongly be supported by a comparison of all seven CSRs (The proposal to establish a novel GH13 subfamily around the cyclomaltodextrinase from ven CSRs  characteven CSRs  in the even CSRs . The treven CSRs . First, ven CSRs . The comven CSRs a that, iven CSRs , containven CSRs b.Flavobacterium sp. No. 92 [Flavobacterium sp. No. 92 cyclomaltodextrinase shares no exceptionally high level of structural similarity with any GH13 subfamily. This fact also supports the independence of the entire group it represents as a novel subfamily.As far as the structural comparison of the full-length cyclomaltodextrinase from . No. 92  with its. No. 92 ,30 could. No. 92 ,76,77,78Flavobacterium sp. No. 92, nor the tertiary structures solved to date have been determined with any α-glucan bound to the domain N [Halothermothrix orenii (In the following part of the structural analysis of the cyclomaltodextrinase, attention was aimed at its N-domain itself. The results from the pair-wise comparisons of this domain with representatives of all 14 relevant SBD CBM families [domain N ,29. It, domain N  typical x orenii . Again, Flavobacterium sp. No. 92. A similar CBM-like domain of amylomaltases from Escherichia coli [Corynebacterium glutamicum [The presented in silico experiments were finally completed by docking trials performed in an effort to demonstrate the eventual binding of α-glucans by the N-terminal domain of the cyclomaltodextrinase from hia coli  and Coryutamicum  from theutamicum . Here, futamicum . AlthougFlavobacterium sp. No. 92 definitively deserves the creation of its own new subfamily within the α-amylase family GH13, the subfamily GH13_46.In any case, based on the present study, the entire group of amylolytic enzymes and hypothetical proteins represented by the cyclomaltodextrinase from


Cancer-related fatigue is a prevalent, debilitating condition that can persist for months or years after treatment. In a single-arm clinical trial, the feasibility and safety of a time-restricted eating (TRE) intervention were evaluated among cancer survivors, and initial estimates of within-person change in cancer-related fatigue were obtained.clinicaltrials.gov in January 2020 (NCT04243512).Participants were 4–60 months post-cancer treatment, were experiencing fatigue , and were not following TRE. TRE entailed limiting all food and beverages to a self-selected 10-h window for 14 days. Participants reported their eating window in a daily diary and completed the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), Brief Fatigue Inventory (BFI), and symptom inventory pre- and post-intervention. This study was pre-registered at p=0.008). Most adhered to TRE; 86.1% ate within a 10-h window at least 80% of the days, and the average eating window was 9.33 ± 1.05 h. Fatigue scores improved 5.3 ± 8.1 points on the FACIT-F fatigue subscale , 30.6 ± 35.9 points for the FACIT-F total score , and −1.0 ± 1.7 points on the BFI .Participants (n=39) were 61.5 ± 12.4 years old and 1.8 ± 1.3 years post-treatment; 89.7% had had breast cancer. The intervention was feasible in that 36/39 (92.3%) of participants completed all questionnaires and daily diaries. It was also safe with no severe adverse events or rapid weight loss (average loss of 1.1 ± 2.3 pounds, A 10-h TRE intervention was feasible and safe among survivors, and fatigue improved with a moderate effect size after two weeks.per se. However, this feasibility trial supports evaluation of TRE in randomized controlled trials to address persistent cancer-related fatigue.This was a single-arm study, so it is possible that expectation effects were present for fatigue outcomes, independent of effects of TRE  Cancer-related fatigue affects at least 30–90% of patients who undergo treatment for cancer treatment, including both chemotherapy and radiation . It is o“Time-restricted eating” refers to restricting eating to a consistent time window between 6 and 12 hours without an overt attempt to reduce caloric intake. Time-restricted eating as a therapeutic approach has garnered substantial appreciation in the literature and among the public in the last decade for its effectiveness to sustain circadian rhythms to prevent and treat disease –13). TheThus, we hypothesize that time-restricted eating can help alleviate persistent cancer-related fatigue. Herein, we performed a two-week, single-arm pilot study that evaluated the feasibility of recruiting cancer survivors to a 10-hour time-restricted eating study, adherence of the participants to the program, and safety of a 10-hour time-restricted eating program in regard to adverse events with a special attention to weight loss. Further, we assessed within-person changes in patient-reported fatigue from pre- to post-intervention.clinicaltrials.gov on 28/01/2020 (NCT04243512). The research protocol was reviewed and approved by the Research Subjects Review Board at URMC (study no. 00004598). All methods were performed in accordance with the Declarations of Helsinki.This prospective single-arm pilot clinical trial was conducted at University of Rochester Medical Center (URMC) from June 2020-September 2021. This study was pre-registered at 2; did not have surgery planned in the next month; did not have any contraindications to the proposed nutrition intervention as identified by their medical provider, their designee, or the study team ; were not taking insulin; and were not taking enteral or parenteral nutrition.Participants were eligible if they had completed adjuvant chemotherapy, surgery, and/or radiation for cancer at least 4 months and not more than 5 years prior to enrolling; had a baseline level of fatigue as determined by reporting a score of 3 or greater for the question, “In the last week, how bad was your worst fatigue on a scale from 0–10?”; spoke English; were at least 18 years old; were not already in the habit of eating all their food within a 10–hour window; had a body mass index (BMI) >20.0 kg/mWhile all participants were recruited locally, all recruitment and study activities were accomplished remotely during the coronavirus disease 2019 (COVID-19) pandemic. Participants were provided the choice to consent via a paper consent (via mail) or eConsent using REDCap software ,16. PartThe restricted eating window was 10 hours long, during the day, and was selected by the participant based on their normal meal patterns and preferences. We asked that the window be consistent during the study period. Water and medications were allowable any time but, because of the potential of caffeine and artificial sweeteners to affect circadian rhythm ,18, coffa priori primary outcome was adherence to time-restricted eating, as calculated from the percent of days that each participant adhered to the intervention.Participants completed a paper-based daily diary for each of the 14 days of the intervention, which included the time of their first calorie and time of their last calorie. The daily eating window was calculated as the difference between first and last calorie. The now as well as the usual and worst in the last 24 hours. It also includes six single-item questions regarding how fatigue has interfered with general activity, mood, work, etc. The average of all 10 items yields a global fatigue score. Cronbach alpha reliability ranges from 0.82 to 0.97 [At baseline and Day 14, a battery of three questionnaires was administered to assess fatigue and other symptoms-the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) , Brief F to 0.97 . Lastly,A 24-hour food record was collected at baseline and Day 14, in which participants were asked to record all food and beverage intake, including time eaten and portion size. These data were entered into NDSR software  and analyzed for total caloric intake.Adverse events were closely monitored as described by the Common Terminology Criteria for Adverse Events (CTCAE), version 5 , with spp<0.05 was deemed statistically significant.Descriptive analyses  were used to describe the study sample demographics, feasibility metrics, and outcomes. A paired t-test was used to assess symptom measures at post- vs. pre-intervention for participants with evaluable data at both time points. Effect size (ES) was calculated as the change in the measure from pre- to post-intervention divided by the pooled SD. A total of 215 letters were sent to potential participants and we had nine referrals from colleagues. We then discussed our study with 59 potential participants . Of thes2), eight (20.5%) were overweight (25 ≤ BMI <30 kg/m2), and 23 (59.0%) were obese (BMI ≥ 30 kg/m2).Participants were 61.5 ± 12.4 years old, 92% female, and 84.8% were White, non-Hispanic . The maja priori minimum of 80% to declare the program feasible. In regard to safety, there were two Grade 1 adverse events—one instance of headache, which was possibly related to the intervention, and one instance of insomnia, which was unlikely to be related to the intervention. Both occurred within the first few days of starting time-restricted eating. There were no other adverse events, including severe adverse events. Participants lost 1.1 ± 2.3 pounds over the course of the study (p=0.008). Based on 24-hour food logs, participants (n=35) consumed 1858 ± 554 kcal at baseline and 1663 ± 462 kcal on Day 14, a difference of 202 ± 654 kcal (10.5 ± 35.2%) fewer total calories (p=0.076).Our primary aim was to assess adherence of the participants to the 10-h time-restricted eating intervention. Daily diaries were collected from 36 participants (92.3%), who completed 100% of entries. On average, participants adhered to the 10-h window 90.1% of the days, and the average eating window was 9 hours, 20 minutes . A total of 86.1% of participants adhered more than 80% of the days, which was larger than our p<0.001, ES=0.50). For the FACIT-F fatigue subscale, fatigue improved by 5.3 ± 8.1 points . The BFI reflected improvements in fatigue, with global scores changing −1.0 ± 1.7 points . Overall, participants enjoyed time-restricted eating and many indicated that it facilitated a more regular eating pattern. Several people reported that it took about three days to one week for their bodies to adjust to the new pattern, after which it was easier and more comfortable. The vast majority voiced that they would continue time-restricted eating in some capacity, some with modifications such as drinking coffee earlier. Some did not like the strictness of the time window but said that they benefited from not eating after dinner and will continue to follow that practice. All participants would recommend time-restricted eating to friends or family, often with specific goals including weight management/loss, regulating blood sugar, reducing fatigue, or promoting overall health. The largest barriers to time-restricted eating included coordinating with their work schedule, coordinating meal times with their family/spouse, and not being allowed to drink caffeinated beverages in the morning. Through this study, some participants reflected on other lifestyle behaviors including diet  and physical activity.Herein, we demonstrated feasibility of a 14-day, 10-hour time-restricted eating intervention among cancer survivors with persistent cancer-related fatigue, as well as initial efficacy. The intervention was very well received, and the vast majority of participants stated that they will continue a time-restricted eating in some fashion. This intervention was safe, and participants reported clinically meaningful improvements in their fatigue using uni- and multidimensional fatigue questionnaires. These results support further testing of a 10-hour time-restricted eating intervention vs. usual care and time- and attention-control interventions to alleviate cancer-related fatigue in cancer survivorship.Adherence was excellent, with 86% adhering at least 80% of the days, and an average eating window of 9 hours, 20 minutes. This is in line with other studies that have evaluated adherence to 8–10-hour time-restricted eating regimens. For example, healthy participants adhered to a 10–12-hour window (self-selected) 6.5 ± 0.5 days/week during a 12-week-long study  . Adults Our results build upon literature showing that cancer survivors with persistent cancer-related fatigue are willing and able to adhere to behavioral interventions to address their fatigue. Currently, behavioral interventions such as exercise and psychosocial interventions  are more effective to combat fatigue than the available pharmaceuticals , and effTime-restricted eating has the potential to improve several pathophysiological mechanisms that underlie cancer-related fatigue. First, time-restricted eating may be able to alleviate fatigue by entraining circadian rhythms. Circadian rhythms are 24-hour biological cycles that work in synchrony to regulate hormone secretion, the sleep/wake cycle, and metabolic processes. Cancer and cancer treatment have been shown to disrupt circadian rhythm, which can lead to sleep disturbances and fatigue –33. CircThe results of this study should take into account both its strengths and limitations. It is one of the first studies testing time-restricted eating in the cancer population, and the first, to our knowledge, to apply it to address chronic fatigue and supportive care outcomes. Our study is limited in that it was small and conducted in mostly older breast cancer survivors, so generalizability should occur with prudence. Also, recruitment occurred during 2020–2021; it is unknown how the COVID-19 pandemic impacted the feasibility of recruitment and participants’ ability to adhere to time-restricted eating. However, because our study was conducted completely remotely, our methods may be used for follow-up multisite studies and studies in rural and other hard-to-reach populations. Lastly, our study was only 14 days long and time-restricted eating interventions that report health benefits are usually at least 8 weeks ,8; it isCancer survivors with persistent cancer-related fatigue were willing and able to adhere to a 10-hour time-restricted eating intervention for two weeks with eagerness to continue the eating pattern. Time-restricted eating was safe and fatigue improved significantly from pre- to post-intervention with moderate effect sizes. These results support future work investigating the effects of time-restricted eating against usual care and other behavioral control interventions to address cancer-related fatigue.


N-carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the Escherichia coli DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines.Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate  B2+-affinity chromatography. Through crystallization screening, the crystals of the huDHOase–malate complex were grown at room temperature through hanging drop vapor diffusion in 0.1 M malate and 1.6 M potassium sodium tartrate tetrahydrate at pH 6.0. For the huDHOase–5-FU complex, the crystals were grown in 0.1 M MES, 1.6 M potassium sodium tartrate tetrahydrate, and 200 μM of 5-FU at pH 6.5.Prior to this study, the complexed crystal structure of huDHOase with either malate or 5-FU was not available. In order to understand whether binding occurs at the active site and investigate the binding mode, the structural information of these huDHOase complexes is needed. As a first step toward determining the binding mode of huDHOase by malate or 5-FU, we attempted to cocrystallize these complexes to further obtain the crystal structures. Recombinant huDHOase (the amino acid residues 1456–1846 in human CAD protein) was overexpressed in E. coli and purified by Ni1, with one molecule per asymmetric unit (N-terminus) and 1822–1846 (the C-terminus) in the ternary structure of huDHOase were disordered and unobserved. The binding of malate  in the huDHOase emission wavelength. Based on this observation, 5-FU was capable of forming a stable complex with huDHOase. As determined through the titration curve, the Kd value of huDHOase bound to 5-FU was 91.2 ± 1.7 μM  were experimentally confirmed  per day, given as continuous intravenous infusion for three weeks [In this study, we identified that huDHOase is capable of interacting with 5-FU, with a ± 1.7 μM . In orde± 1.7 μM . The two± 1.7 μM . In comp.1 ± 1.4  and 133.ee weeks , these rGiven the similarities in their active sites, the 5-FU binding mode of huDHOase might be considered identical to that of ScDHOase . HoweverRegarding the complexed crystal structures of EcDHOase  and huDHIn this study, we found only two metal ions, rather than the three shown in apo-huDHOase , within The crystal structures of HYDase , DHPase In this study, we identified that huDHOase is a novel 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. Mutational and structural analyses indicated that the 5-FU binding mode of huDHOase differed from ScDHOase and PaDHPase. Considering the loop-in mode revealed by our structures complexed with huDHOase, the dynamic loop in DHOase should be a suitable drug target for inhibiting pyrimidine biosynthesis. This complex structure might provide insights into how 5-FU and its pyrimidine derivatives could bind to and inhibit the proteins in cancer-signaling pathways. We also found a potential interface for the dimerization of huDHOase. The subunit-interacting residues of huDHOase for dimerization are significantly different from other DHOases. Given that microbiota can modulate the host response to 5-FU, further research should directly focus on revisiting the role of bacterial and human DHOase in anticancer therapy.